Use of 13C glucose in an analytical assay to monitor glucose metabolism by measurement of labeled exhaled CO2 is provided. A breath test and kit for performing the breath test are described for the diagnosis of diabetic indications and monitoring of glycemic control. The breath test utilizes the measurement of expired 13C-labeled CO2 following the ingestion of a 13C-enriched glucose source.
The following references are referred to by their numbers in parenthesis in this specification.
1 Martin B C, Warram J H, Krolewski A S, et al. Role of glucose and insulin resistance in development of type 2 diabetes mellitus: results of a 25-year follow-up study. Lancet 1992; 340: 925-9.
2. Lillioja S, Mott D M, Spraul M, et al. Insulin resistance and insulin secretory dysfunction as precursors of non-insulin-dependent diabetes mellitus; prospective studies of Pima Indians. N Engl J Med 1993; 329: 1988-92.
3. Beck Nielsen H, Groop L C. Metabolic and genetic characterization of pre-diabetic states. Sequence of events leading to non-insulin-dependent diabetes mellitus. J Clin Invest 1994; 94: 1714-21.
4. Matthaei S, Stumvoll M, Kellerer M, et al. Pathophysiology and pharmacological treatment of insulin resistance. Endocr Rev 2000; 21: 585-618.
5. The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus. Report of the expert committee on the diagnosis and classification of diabetes mellitus. Diabetes Care 2001; 24(suppl 1).
6. Harris M I. Undiagnosed NIDDM: clinical and public health issues. Diabetes Care 1993, 16: 642-52.
7. World Health Organization. Prevention of diabetes mellitus: report of a WHO study group. Geneva: WHO, 1994; technical report series No. 844.
8. Meltzer S, Leiter L, Daneman D, et al. 1998 clinical practice guidelines for the management of diabetes in Canada. Can Med Assoc J 1998; 159 (suppl 8): S1-29.
9. Matthews D R, Hosker J P, Rudenski A S, et al. Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man. Diabetologia 1985; 28: 412-19.
10. Haffner S M, Gonzales C, Miettinene H, et al. A prospective analysis of the HOMA model: the Mexico City Diabetes Study. Diabetes Care 1996: 19: 1138-41.
11. Bonora E, Targher G, Alberiche M, et al. Homeostasis model assessment closely mirrors the glucose clamp technique in the assessment of insulin sensitivity. Diabetes Care 2000; 23: 57-63.
12. World Health Organization. Definition, diagnosis and classification of diabetes mellitus and its complications: Report of a WHO Consultation. Part 1. Diagnosis and classification of diabetes mellitus. Geneva: WHO, 1999.
13. Ganda O P, Day J L, Soeldner J S, et al. Reproducibility and comparative analysis of repeated intravenous and oral glucose tolerance tests. Diabetes 1978; 27:715-25.
14. Riccardi G, Vaccaro O, Rivellese A, et al. Reproducibility of the new diagnostic criteria for impaired glucose tolerance. Am J Epidemiol 1985; 121: 422-9.
15. Ko GTC, Chan JCN, Woo J, et al. Use of the 1997 American Diabetes Association Diagnostic criteria for diabetes in a Hong Kong Chinese population. Diabetes Care 1998; 21: 2094-7.
16. Ko GTC, Chan J C N, Woo J, et al. The reproducibility and usefulness of the oral glucose tolerance test in screening for diabetes and other cardiovascular risk factors. Ann Clin Biochem 1998; 35: 62-7.
17. Gabir M M, Hanson R L, Diabelea D, et al. The 1997 American Diabetes Association and 1999 World Health Organization criteria for hyperglycemia in the diagnosis and prediction of diabetes Diabetes Care 2000; 23: 1108-12.
18. Radziuk J. Insulin sensitivity and its measurement: structural commonalities among the methods. J Clin Endocrinol Metab 2000; 85: 4426-33.
19. CDC Diabetes Cost-Effectiveness Study Group. The cost-effectiveness of screening for type 2 diabetes. JAMA 1998; 280: 1757-63.
20. Hosker J P, Matthews D R, Rudneski A S, et al. Continuous infusion of glucose with model assessment: measurement of insulin resistance and xcex2-cell function in man. Diabetologia 1985; 28: 401-11.
21. Emoto M, Kawagishi T, Nishizawa Y, et al. Homeostasis model assessment as a clinical index of insulin resistance in type 2 diabetic patients treated with sulfonylureas. Diabetes Care 1999; 22:818-22.
The entire disclosure of each of the above-referenced publications, patents and patent applications is incorporated herein in its entirety.
Glucose tolerance is defined as the ability to properly utilize glucose. Diabetes is not a single disease, but an array of diseases that exhibit the common symptom of glucose intolerance, an impairment in glucose utilization.
The prevalence of diabetes in the general population is approximately 6-7%. Only about half of diabetics are actually diagnosed. Studies have shown that rates for persons with glucose intolerance are equal by sex and greater for blacks than for whites.
In general, the following types of diabetes have been recognized: type I diabetes mellitus, type II diabetes mellitus, secondary diabetes mellitus, impaired glucose tolerance and gestational glucose mellitus. The general characteristics of the symptoms of diabetes include the following:
Polyuria (excretion of large quantities of urine)
Hyperglycemia (high blood glucose levels)
Glucosuria (abnormal presence of glucose in urine)
Polydipsia (excessive thirst)
Polyphagia (excessive hunger)
Sudden weight loss
It has been observed that complications resulting from diabetes mellitus are the third leading cause of death in most developed countries. Diabetes is a risk factor for a variety of conditions including coronary heart disease, cerebrovascular stroke, neuropathy (nerve damage), nephropathy (kidney damage), retinopathy (eye damage), hyperlipidemia (excessive blood lipids), angiopathy (damage to blood vessels) and infection.
A number of different methods exist for determining a condition of intolerance for glucose. These include postprandial blood glucose, oral glucose tolerance test (OGTT), O""Sullivan glucose tolerance test (gestational test), hemoglobin Alc (Hb A1, Hb A1c), islet cell antibodies, glutamic acid decarboxylase (GAD) antibodies and insulin antibodies. Diabetes, however, is most readily detected when the carbohydrate metabolic capacity is tested. This is done by stressing the system with a defined glucose load as in the oral glucose tolerance test (OGTT).
The OGTT has been criticized, however, because many of the variables affecting test results are difficult to control. For instance: patients must be on a standardized carbohydrate diet at least three days before the test; the test requires an 8 to 16 hour fast; the test should only be performed on ambulatory patients; stress should be avoided; exercise should be avoided; various hormone imbalances can affect validity such as with: thyroxine, growth hormone, cortisol and catecholamines; various drugs and medications can affect validity such as: oral contraceptives, salicylates, nicotinic acid, diuretics and hypoglycemics; and evaluation should normally be corrected for age. The greatest disadvantage of the OGTT is that it is poorly reproducible and this limits its diagnostic usefulness.
Type 2 diabetes is a common condition, associated with significant morbidity and mortality. It is generally acknowledged that overt type 2 diabetes is preceded by a period of glucose intolerance which itself is preceded by a significant period of insulin resistance (1-5). It is now further recognized that typical diabetic complications can begin to develop during this xe2x80x9cpre-diabeticxe2x80x9d phase (3, 6). The identification of persons at risk of developing overt type 2 diabetes has therefore taken on even greater importance. It has been suggested that if such persons could be easily identified, a lifestyle modification strategy could be implemented which might prevent their progression to type 2 diabetes with its attendant morbidities.
Because of the public health importance of type 2 diabetes, regular screening for this condition is now advocated (5, 7, 8). However, such screening programs, whether by fasting plasma glucose or by the 75-g OGTT, only identify diabetic or glucose-intolerant patients. The homeostasis model assessment (HOMA) index has been advocated as a method of detecting persons with insulin resistance and therefore presumably at risk of progressing to overt type 2 diabetes (9-11). However, the HOMA index requires a serum insulin measurement and, some argue, the use of a computer program. Thus, this index is not as simple or accessible as a fasting blood glucose level. Similarly, the gold standard euglycemic, hyperinsulinemic clamp is clearly not appropriate for mass screening campaigns.
The current methods of diagnosing diabetes involve either invasive testing (i.e., repeated blood collections), or use blood-borne markers (i.e., glycosylated proteins, or antibodies) which offer an indirect assessment of glucose regulation. Accordingly, it is an object of the present invention to avoid the need for invasive testing or the use of blood-borne markers in determinations of glucose regulation.
The above and other objects of the invention are attained by a 13C breath test and a kit for determining glucose regulation in a patient in need thereof.
Based on our experience in the use of 13C breath tests, we propose a simple, sensitive test of insulin resistance. In normal individuals, in the presence of insulin, glucose is taken up by cells where it undergoes glycolysis and then enters the citric acid cycle or is shunted to fat synthesis. In either case, CO2 is produced as a metabolic by-product. This CO2 then re-enters the circulation and is eliminated in the lungs. We found that if glucose was labeled with 13C, the resultant CO2 could be detected in the expired air. In type 2 diabetes and other states of insulin resistance, glucose uptake is impaired and the generation of 13Co2 is likewise blunted. Accordingly, we have developed a 13C-glucose breath test for the diagnosis of type 2 diabetes and insulin resistance. In particular, the test provides a means to detect insulin resistance when blood glucose levels are still in the normal range and before xcex2-cell destruction leading to diabetes has occurred. Early detection of insulin resistance will allow intervention in time to prevent the development of type 2 diabetes. In addition, the test allows the success of intervention therapies, including diet and exercise. to be monitored.
An analytical assay is described that is based on the use of non-radioactive 13C. Labeled expired 13CO2 is measured in the present assay. Isotope ratio mass spectroscopy (IRMS) is used as a detection method for 13C, a non-radioactive isotope that occurs naturally in food and animal tissues. Non-dispersive infrared spectroscopy (NDIRS) analysis and analysis methods known in the art may be employed. The test protocol is as follows: after an overnight fast, the oral dose of 13C uniformly labeled glucose (containing about 25 mg of 13C glucose in combination with about 15 g of unlabeled glucose in 100 ml of tap water) is administered. Breath samples will be collected before the dose and then 1xc2xd hours after 13C glucose ingestion. Levels of 13CO2 in expired air will be measured by an IRMS method.
Advantages of this test are the following:
it is practical, sensitive and specific;
the validity of the test is not influenced by stress, exercise, hormone imbalances, or some drugs and medications;
it is a non-invasive method;
it is simple to perform and can be readily used in physicians"" offices or medical laboratories;
it is safe since 13C is a naturally occurring isotope found in all carbon-containing substances;
it involves no radioactivity, and may be used in children and women.
The 13C glucose test is safe, reliable, and specific in diagnosis of diabetes and measurement of the severity of insulin resistance in patients. The invention is also preferred to diagnose gestational diabetes and to monitor glycemic control in diabetes patients. A preferred embodiment of the invention is a kit containing the necessary material for performing the described method. This kit may contain, but is not limited to, a source of 13C enriched glucose (preferably uniformly labeled D-glucose); a source of unenriched glucose; and a breath collection device. The kit may also contain a set of patient instructions for its use. In another embodiment, the kit may additionally contain a blood collection device, such as a lancet or hypodermic needle and vacutainer for the additional determination of blood glucose levels.
Accordingly, in one aspect the invention provides diagnostic kits for the determination of glycemic control in a subject comprising: a predetermined quantity of 13C-enriched glucose; and a breath collection container. A plurality of breath containers and/or instructions for use may be included. The kits may be used for the diagnosis of diabetes, insulin resistance, gestational diabetes, and the like or to determine the adequacy of antihyperglycemic therapy.
In a further aspect, the invention provides a use of 13C-enriched glucose for the determination of glycemic control in a subject.
In another aspect, the invention provides 13C-enriched glucose for use in the manufacture of diagnostic kits for the determination of glycemic control in a subject. The kits may be used for the diagnosis of diabetes, insulin resistance, gestational diabetes, and the like or to determine the adequacy of antihyperglycemic therapy.
In yet a further aspect, the invention provides diagnostic kits for the determination of glycemic control in normal, diabetic and insulin resistant subjects by comparing blood glucose levels with breath levels of 13C-enriched CO2 
In a still further aspect, the invention provides method of diagnosing a condition in a subject, said condition selected from the group consisting of diabetes, insulin resistance impaired glucose tolerance, impaired fasting glucose and gestational diabetes, said method comprising collecting a first breath sample from said subject in a first breath collection container; administering 13C-enriched glucose to said subject; collecting a second breath sample from said subject in a second breath container at a time point after administration of said 13C-enriched glucose; measuring the 13CO2 in each of said first and second breath samples; and comparing the amount of 13CO2 in said second breath sample with the amount of 13CO2 in said first breath sample to obtain a delta value, wherein the presence of less 13CO2 in said second breath sample compared to normal control values indicates the presence of said condition. Using an ROC curve, a delta cutoff is chosen wherein the sensitivity and specificity are such as to maximize diagnostic accuracy. In particular, when the condition is insulin resistance, a range of deltas from 8 to 10 is preferred. A delta of 9 is most preferred.
In yet an additional aspect, the invention provides method of predicting a subject""s risk of developing diabetes, said method comprising collecting a first breath sample from said subject in a first breath collection container; administering 3C-enriched glucose to said subject; collecting a second breath sample from said subject in a second breath container at a time point after administration of said 13C-enriched glucose; measuring the 13CO2 in each of said first and second breath samples; and comparing the amount of 13CO2 in said second breath sample with the amount of 13CO2 in said first breath sample, wherein the presence of less 13CO2 in said second breath sample compared to normal control values indicates risk of developing diabetes. The comparison may be made by choosing a cutoff of ROC values wherein the sensitivity and specificity are such as to maximize diagnostic accuracy. In particular, a range of ROC""s from 8 to 10 is preferred. An ROC of 9 is most preferred.
The 13C-glucose breath test is superior to currently used laboratory criteria in the diagnosis of type 2 diabetes. Its predictive value for clinical status, as well as its correlation with the HOMA index, make it a simple but useful test for detecting early evidence of insulin resistance and hence, risk for type 2 diabetes.